Figure 6. TCF1 knockdown in PyMT cells attenuates sphere formation through Cyclin D1 downregulation.

(A) PyMT1 and p21KO1 cell lines that were untreated or treated with Wnt3a for 18 hrs were assayed for TCF1 mRNA expression by Taqman qRT-PCR. Significant differences in TCF1 expression (p<0.05) were noted between untreated or Wnt3a-treated PyMT1 and p21KO1 cell lines. (B) PyMT1 cells expressing control shRNA, TCF1shRNA1 or TCF1shRNA2, were untreated or treated with Wnt3a for 18 hrs, and tested for TCF1 mRNA expression, or (C) assayed for TOP/FOP Flash activity in the absence or presence of 10μM ICG001. Significant differences were seen between PyMT1 and PyMT/TCF1shRNA1 and PyMT/TCF1shRNA2; p<0.05. (D) PyMT1 were compared to PyMT1/TCF1shRNA1 (sh1) and PyMT1/TCF1shRNA2 (sh2) for mammosphere formation. The percent of spheres in triplicate experiments is shown as mean ± SEM, p<0.05. (E) PyMT1, PyMT1/TCF1shRNA1 and PyMT1/TCF1shRNA2 cells were assayed for levels of Cyclin D1 using qRT-PCR. Significant differences in Cyclin D1 mRNA level were noted between PyMT1 and TCF1 knockdown cells; p<0.05. (F) PyMT1 cells were treated with control shRNA (lane 1) and 4 Cyclin D1 shRNAs; levels of Cyclin D1 assessed by western blotting. Note shRNA 3 and 4 were most efficient in knocking down Cyclin D1 expression. PyMT1 expressing control and CyclinD1 shRNA3 and shRNA4 were assayed for mammosphere formation. The percentage of spheres was measured in triplicates, showing significant differences between PyMT1 and Cyclin D1 knockdown cultures (p<0.05).